The main objective of the parent Phase II SBIR grant is to develop our novel NGS sequencing library platform, miR- SEQ, as a ready-to-use kit that allows profiling of cell-free (cf)-miRNAs in plasma samples without the need to extract RNA. Profiling of extracellular miRNAs from biofluids is important for the discovery of biomarkers indicative of, for example, disease status and efficacy of treatment [1]. Recent reports confirm that cf-miRNA expression among healthy subjects is highly variable, suggesting that a large number of samples are required to identify statistically significant changes in expression above random noise [1, 2]. Based on these reports as well as our own previous data, it is clear that accomplishing the aims of our grant will require a large number of samples, probably more than was originally estimated. In addition to the individual variability among subjects, the levels of individual miRNAs circulating in plasma span a wide range of concentrations [2], requiring a wide dynamic range of detection. To maximize the dynamic range of miR-SEQ and thereby expand the number of miRNAs that can be accurately detected, we separately profile clusters of miRNAs with similar expression levels. This approach requires a higher sequencing coverage than a no-clustering approach. Since the aims of the parent miR-SEQ grant require both a large number of samples and high sequencing coverage, we had planned to use a HiSeq instrument to conduct the work. Originally, through a collaborative agreement with DoveTail Genomics, another company located in our building, we had unrestricted ?at cost? access to DoveTail?s HiSeq sequencer. This ensured a fast turnaround time with minimal down-time between sequencing runs so that the research aims could be efficiently achieved within the grant?s allotted timeframe. Unfortunately, we were recently notified that Dovetail is relocating shortly, unexpectedly ending our collaborative agreement and curtailing our access to the HiSeq instrument. As we are only in Year 1 of the 3-year grant, it is critical that we obtain access to another medium-to-high- throughput sequencer. Although several commercial vendors offer sequencing services, their slow turnaround times makes this alternative very problematic. In our experience it is not uncommon for the turnaround time from commercial vendors to be upwards of 1 month between runs. Such a turnaround-time will significantly slow progress and impact our ability to complete the aims in this grant, as the design of each experiment depends on the sequencing results of the previous experiment. Therefore, there will be limited work conducted on the project in this interim. There are also logistical and sample-handling problems with shipping the volume and complexity of samples necessary to complete the aims of this grant. Also, since much of the work will be conducted using clinical samples provided by outside collaborators, we are bound by certain sample use and transfer restrictions imposed by our collaborators? institutions. These restrictions impose additional difficulties, making sending material to third party vendors much more difficult if not impossible in some instances. Integral to completion of our grant Aims is the work of two active collaborations to profile miRNA biomarkers, which also requires access to the appropriate sequencing equipment: 1) With Prof. Susan Carpenter from the University of California Santa Cruz (letter of support attached) we aim to identify circulating biomarkers of early-stage Rheumatoid Arthritis. In the initial phase of this grant we are profiling by non-targeted sequencing a small subset of samples with the goal of identifying all miRNAs and other small RNAs present in serum samples from RA patients and matching controls. This preliminary work provides the basis for development of the targeted sequencing approach that will profile miRNAs from a sample size of at least 100 patients and 100 matching controls. 2) With Prof. Lakis Liloglou from the University of Liverpool (letter of support attached), we aim to show that targeted sequencing can identify miRNAs present in specific vesicles (e.g., exosomes), as well as in whole plasma. This collaboration will initially profile miRNAs from either complete plasma or from isolated exosomes to identify lung cancer biomarkers and conclude which biomarkers are most abundant. These results will be used to identify appropriate candidates for targeted panels. In the second phase of this collaboration we will profile a larger set of samples from the Liverpool Lung Project by targeted sequencing, to further characterize lung cancer miRNA biomarkers. These collaborations are an integral part of our grant and should provide data for three different aims. They will be used as part of Aim 3: Expand set of probes targeting plasma miRNAs (specifically Task 3.2), that aims to profile plasma samples to high sequencing coverage to detect novel plasma miRNAs. Profiling of these samples will allow us to design probes to target all circulating miRNAs and proceed with the rest of the aims. Samples from both collaborations will be also used as part of Aim 5, to benchmark our technology against other targeted sequencing approaches; and for Aim 6 to profile plasma samples from healthy donors and patients to determine the sensitivity of our technology in identifying miRNA signatures of particular diseases. Considering the complexity of sample management, the sequencing throughput required, turnaround time limitations, and the active collaborations required to fulfill the aims of this grant, we wish to request additional funds to obtain our own medium-throughput sequencer. Our research of the options suggests that a medium-throughput sequencer such as the NextSeq instrument is the most cost-effective choice for our needs. We have obtained a quote from Illumina for a NextSeq instrument (see specs in Figure 1 and attached quote). This instrument allows the generation of 400 million reads in each single run with the high-output reagents, all in under 12 hours. Considering that a coverage of 10-20 million reads per sample is required to detect the rarest of circulating miRNAs, this instrument will allow the sequencing of up to 20 samples per run. This fast and medium-throughput sequencing capability will allow us to achieve the turnaround time, sample throughput and sequencing depth required to finish the aims proposed for the present grant.